Autoreactive T cells are thought to play a critical role in the pathogenesis of multiple sclerosis (MS), yet the functional T cell programs that promote disease remain unknown. A major challenge hindering the study of self-reactive T cells in MS is the sensitive identification and characterization of these cells. The technologies used to date have insufficient sensitivity for detection of T cells with low TCR affinities and may bias detection for cells with sufficient in vitro proliferative potential. To overcome these challenges, this collaborative project brings together the Love lab (MiT) with expertise in simultaneous parallel phenotypic and functional analyses of >10^ single primary cells in arrays of sub-nanoliter wells (nanowells) and the Hafler lab (Yale School of Medicine) with expertise in characterization of autoreactive T cells in MS as well as the genetics of autoimmune disease. Specifically, this project will address a central question related to understanding the pathology of MS: Po patients with MS have increased numbers of myelin-reactive IL- 17/GM-CSF/IFN?-secreting CP4+CCR6+CP45RO+ cells compared to age-matched control subjects? To address this critical question, a two-pronged approach will be employed that leverages a new method for polyclonal expansion of CP4+ T cells in small pools, and a novel state-of-the-art nanowell technology for single-cell co-culture assays of individual T cells with autologous mature dendritic cells pulsed with antigen. This technology enables sensitive detection of self-reactive T cells and generates a comprehensive body of data on surface phenotype and cytokine release using a dense, elastomeric array of nanowells. Many different cytokines are captured on a glass slide and quantified on a microarray scanner. Preliminary data show that this approach greatly increases the sensitivity of detection for self-reactive T cells and enables comprehensive assessment of their ex vivo functions. As a second aspect of this aim, we will examine how genotypic variations in HLA haplotypes and cumulative genetic loads in the IL-17 pathway affect the burden of IL- 17/GM-CSF/IFN?+ T cells. In Aim 2, we will address how distinct populations of antigen presenting cells (APCs) alter the functional programs of myelin-reactive T cells from blood and cerebrospinal fluid (CSF). Of particular interest is the interaction of podoplanin (PPPN) expressed by CSF T cells with CLEC-2 on B cells/dendritic cells, given the importance of PPPN in the formation of ectopic lymphoid follicles in the CNS of patients with MS. The outcome of these studies will provide new insight into the ex wVo function of myelinreactive T cells, and the impact of distinct APCs on their functional programs.